There is intense interest in the circuits that guide stem cell behavior. While niches are essential to the behavior of many tissue-specific stem cells, it is not understood how the niche is specified and assembled in a tissue, and then how it executes control over the stem cell pool. Understanding these interactions will be crucial to use these cells in regenerative medicine. This proposal addresses how are niches specified, organized and function, and utilizes one of the most well-understood stem cell-niche systems, the Drosophila testis. Here, a small group of cells (hub cells) act as part of the niche, leading t the activation of signaling pathways in adjacent cells. In this way, nearby somatic cells take on cyst stem cell fate (CySC), while nearby germline cells, intermingled with these CySCs, take on germline stem cell fate (GSC). Hub formation, and the attendant attachment of stem cells, is the major architectural event of gonadogenesis. The specification and placement of hub cells among somatic gonadal precursors (SGPs) generates an anteriorly-anchored proliferation center that will drive spermatogenesis in a polarized manner. To generate that polarity, a subset of pre-hub cells migrates through the germ cell milieu of the forming gonad, and undergoes a mesenchymal-to-epithelial transition (MET), only then acting as niche cells. Finally, the key self-renewal signal is delivered by BMPs expressed from both hub cells and CySCs. The first Aim uses a combination of live-imaging and loss- and gain-of-function studies to explore cytoskeletal control of pre-hub cell migration, and the mesenchymal-to-epithelial transition necessary for niche formation. A second Aim focuses on Zfh1, a transcriptional regulator which is key to CySC self- renewal and to how CySCs act as niche cells for GSC renewal. Targets of Zfh1 will be indentified and analyzed functionally. This will define genes important for CySC self- renewal, for the production of renewal signals for GSCs, and for the control of MET.